Our long-term goals are to develop a detailed mechanistic understanding of HIV-1 proteins using large-scale mutagenesis to probe structure/function relationships. We have developed technologies that have allowed us to make the HIV-1 protease one of the most thoroughly characterized proteins analyzed by genetic techniques. The continued application of these techniques will provide an important source of information that can be combined with other approaches, such as biochemical and structural analysis, to yield important insight into key enzymes in the viral replication cycle. The specific focus of the proposed research is to carry out a detailed genetic analysis of several of the HIV-1 pol gene products: protease, reverse transcriptase, and RNase H. The basis for this work is our previous mutational mapping of the viral protease in which we characterized mutants with substitutions at each amino acid of the protease. We will continue to define the sequence requirements for protease activity and search for novel or conditional mutant phenotypes that give insight into the role of specific amino acids in protease function. We will also characterize the processing pathway in the maturation of virions, and use mutagenesis to define the sequence requirements of the protease cleavage sites placed within the natural substrate. The role of the protease in cell killing will also be explored. We will extend our genetic analysis to the reverse transcriptase and RNase H activities of HIV-1. Through large-scale mutagenesis, functionally important domains of these proteins will be identified and novel phenotypes uncovered. These studies will provide important information about the function organization of these two poorly understood enzymes.